A major focus of pharmaceutical research has concentrated on developing molecular entities to regulate gene expression. It is believed that modulation of specific genes will alter or circumvent molecular mechanisms underlying acute and chronic disease.
Many approved antineoplastic agents demonstrate significant adverse effects. Newer therapies seek to avoid non-specific toxicities by selectively targeting cell and disease-specific pathways associated with growth, senescence and cellular transformation.
Highly proliferating cells and groups thereof are often the product of multiple events. With time and unchecked by normal processes maintaining homeostasis, proliferating cells expand in number where some may become genetically unstable and diverse. With continued growth, probabilities increase that cells within these groups acquire and express traits enabling escape from or adaptation to therapy. These cells and groups thereof with time will not possess a single target or Achilles' heel through which a mono-functional entity or “magic bullet” could achieve complete therapeutic success. When complex intracellular networks, crosstalk and cellular redundancies that maintain hyperproliferative states are considered, the discovery and development of mono-functional agents may not represent the best or only therapeutic option.
Polynucleic acid-based structures when designed to mimic and interact with two or more cellular features will be effective therapeutic agents. Reflecting on progress and development of antineoplastic therapeutics over the past decades, there remains a need in the art for multifunctional therapeutics. The characterization and design of such structures is provided herein with evidence that these agents are of enhanced utility.
Factors Modulating Genetic Expression
Decoys in context of the invention are polynucleic acid mimetics capable of diverting transcription factors and other proteins from endogenous targets to modulate expression and alter cellular biochemistry. Mimicry is essential to decoys, where cellular aspects, elements or features are resembled, copied closely or imitated accurately.
Nucleic acid structures functioning as transcription factor decoys have been described by Chu and Orgel in U.S. Pat. No. 5,683,985. In this fashion, transcription factors are attractive targets for therapeutic intervention (Mann, M J, et al., 2000, J. Clin. Invest 106, 1071-1075; Morishita, R2003, Curr. Drug Targets. 4, 2-). Bielinska and coworkers described the use of such decoys as tools to alter transcription activity in vitro (Bielinska, A, et al., 1990, Science 250, 997-1000).
Transcription factor decoys show promise in the treatment of neoplastic disease. U.S. Pat. No. 6,060,310 describes CRE-elements with utility in models of tumor cell growth in vitro and in xenograft models. E2F-elements in U.S. Pat. No. 6,774,118 and US Patent Publication Nos. 20020052333, 20030186922, 20030022870 suggest decoys may have utility in models of cardiovascular disease. However, recent human clinical trial data suggests E2F decoys may not be effective as single agents for some cardiovascular indications. U.S. Pat. No. 6,034,234 describes basic E2F-elements 5′-TTTSSCGS-3′ (where S═C or G) that inhibit expression of growth-related genes with broad indications.
GC-rich elements capable of binding SP-factors can act as angiogenesis inhibitors and provide potential therapy in tumor models. Additionally, U.S. Pat. No. 6,262,033 and U.S. Patent Publication Nos. 20020098162 and 20040109843 describe NFKB-elements with utility in diseases associated with cellular proliferation. U.S. Patent Publication No. 20050037494 suggests STAT1 may bind specifically to unique sequences. As constructed and in conjunction with some STAT-elements, ETS-elements may play important roles in angiogenesis, matrix remodeling, and cancer progression.
Decoy approaches to EGR1 will be beneficial as overexpression of EGR1 and target genes have been shown as factors contributing to prostate cell proliferation. Decoys with E-box-elements may also be beneficial as associated with genetic regulation through the binding HIF1A, TFE3, USF1 and other proteins. Of particular importance are MXD1(MAD), and MXI1 as MAX-interacting proteins where deregulation of MYC has been implicated in the development of several human cancers (Hermeking, H2003, Curr. Cancer Drug Targets. 3, 163-175).
Transcription factors and other DNA associating factors as overexpressed in disease states suggest that the use of DNA mimicry will have utility. E-Box-, NFKB-, CRE/ATF- and E2F-related transcription factors are disproportionately overexpressed in specific cancers (Rhodes, D R, et al., 2005, Nat. Genet. 37, 579-583). Particularly attractive towards development of polynucleic acids containing E2F-, SP-, NFKB-, EGR-, E-box-, ETS-, and CRE/ATF-related elements will be activity as angiogenesis inhibitors. Those familiar with current therapeutic strategies for cancers recognize that targeting either active proliferating cells or the neovascularization required for sustaining and facilitating tumor growth as important and desirable characteristics.
Hybridization with cellular polynucleic acids may also be a form of polynucleic acid therapy. ‘Antisense agents’ represent a class of therapeutics designed to inhibit the expression of genes in a selective and sequence specific manner. U.S. Pat. No. 6,015,892 describes the antineoplastic potential of a number of 20 base pair sequences. Antisense methods for PRKCA continues in clinical trials with specific applications in oncology although single agent trials have been discouraging. This provides additional evidence that composite therapeutics or uses in combination with existing chemotherapy are necessary for clinical utility in humans. RAD51C is one homolog of the RAD-family of proteins that have a role in homologous recombination, DNA repair, and cellular proliferation or growth. Increased sensitivity to radiation can also be achieved by antisense methods and amplification of the chromosomal region 17q22-q24 associated with RAD51C/RAD51L3(RAD51D) and RPS6KB1 are common findings in breast cancer that may contribute to an aggressive clinical course.
In mammalian genomic DNA, 5-methylcytosine is the only known modified base where DNA methyltransferases and methylated-DNA-binding proteins are thought to play important biological roles in development and pathological processes. DNMTs may be important targets for therapy (Szyf, M2001, Front Biosci. 6, D599-D609). One attractive therapeutic aspect of DNA methyltransferase inhibitors are that they appear to inhibit DNA replication. U.S. Pat. No. 6,268,137 describes specific DNA hairpin inhibitors of DNA methyltransferase that form stable noncovalent complexes in a manner independent of S-adenosylmethionine. U.S. Pat. No. 5,503,975 describes additional self-associating artificial polynucleic acids that included 5-fluorocytosine (5FC) at positions corresponding to methylation sites. 5-methylcytosine and other modified bases as present in specific CpG containing sequences will also have potential to interact with methylated-DNA-binding proteins.
Characterized with polynucleic acid therapies are additional phenomena associated with immune system modulation. This occurs to varying degrees in response to polynucleotides where 5′-CpG-3′ pairs are present in specific sequence contexts. While there is a great deal of research in attempting to understand CpG molecular effects, a fair generalization is that these represent innate host defense responses to the presence of foreign DNA that can occur with prokaryotic or viral infections. U.S. Pat. No. 6,207,646 is one of many related patents describing these effects. How CpGs present may or may not show immunomodulatory activity, directly or indirectly in concordance with elements for CRE/ATF-, SP-, EGR-, STAT-, E-box-, ETS-, NFKB-elements is unknown as these responses require many of these transcription factors and associated pathways.
Factors Associated with Genetic Integrity
Specific sequences may interfere with proteins associated with telomere maintenance. Inhibitors may be molecular entities that simply resemble ‘telomeres’ or otherwise associate with proteins or polynucleic acid components of proteins involved in telomere maintenance. The human telomeric protein POT1 is known to bind single-stranded DNA and may participate as a regulator of telomere length in association with factors such as TERT and TERC. Treatment with polynucleotides homologous to telomere overhangs may induce senescence with benefits in neoplastic disease (Li, G Z, et al., 2004, Exp. Cell Res. 301, 189-200; Li, GZ, et al., 2003, Proc. Natl. Acad. Sci. U.S.A 100, 527-531). However ‘T-oligo’ treatment alone may not be clinically useful in patients with late stage melanomas again suggesting mono-functional therapies may not be the best clinical approach.
Structures that mimic DNA damage such as gaps, nicks, breaks, or that use non-standard bases and spacers may inhibit cellular growth through multiple pathways associated with DNA repair and genetic integrity that can trigger cellular senescence (e.g., programmed cell death or apoptosis). DNA damage is known to activate checkpoint pathways and halt cell cycle progression. For example, 9-1-1 complexes can be associated with early checkpoint signaling, whereas CDC25A, ATR, CHEK1, and polymerases such as POLB are important for cell cycle progression. DEK may bind more complex polynucleic acid structures independent of sequence. Chromatin-associated poly(ADP-ribose) polymerases (PARPs) may also increase cellular sensitivity to DNA-damaging agents, topoisomerases, and ionizing radiation (Virag, L, et al., 2002, Pharmacol. Rev. 54, 375-429).
Overall, composite agents integrating two or more features inhibiting cellular growth by interfering with genetic expression and triggering pathways related to programmed cell death is intended to create therapeutics of greater efficacy.
Although combinations of polynucleic acid therapies with other small molecules are well known, inter- and intra-associating polynucleic acids integrating two or more features for therapeutic benefit are unrealized or less common. U.S. Patent Publication No. 20040109843 describes the use of a single polynucleic acid targeting NFKB- and ETS-related factors. However, the current invention employs several additional factors without use of intervening sequences. Furthermore, the current invention positions described elements deliberately to impart competitive aspects to binding. Considering the juxtaposition of auxiliary elements, binding of factors as associated with the regulation of genetic expression can be considered ‘either A or B’ not ‘A and B’ characteristic of simple combinations.
The use of a single polynucleic acid sequence complementary to two cellular mRNAs acting as a ‘bispecific antisense agent’ is known. U.S. Patent Publication No. 20030158143 describes the administration of such a bispecific antisense oligonucleotide in amounts effective to reduce IGFBP-2 and/or IGFBP-5 in cells associated with endocrine-regulated tumor cells. Additional bispecific agents are known, however use of one, two or more base or nucleobase sequences that in single stranded form may inhibit cellular growth through complementary hybridization to mRNAs via antisense mechanisms (i.e., PRKCA and RAD51C), that in combination or when used with appropriate complement sequences forms duplexes containing two or more binding sequences for two or more transcription factors is unknown.
Additional multifunctional approaches using polynucleic acid based therapeutics are known. U.S. Patent Publication No. 20050064407 describes a DNAzyme that specifically cleaves up to seven BCL2-related mRNA transcripts with potential utility in the treatment of tumors. This is related to the current invention as an example or therapeutic approach using a multitasking or promiscuous agent (Arteaga, CL2003, Clin. Cancer Res. 9, 1231-1232).
The invention can be further distinguished from prior art in numerous ways. Use of polynucleic acids designed to contain transcription factor binding sites also containing 3′ tailing sequences intended to interact with additional proteins appears unknown. The addition of one or more 3′ overhanging sequences to any duplex-forming sequence capable of functioning as two or more transcription factor decoy(s) for the purpose of also targeting, engaging or interacting with DNA repair proteins or proteins associated with telomere maintenance is unknown.
The use of a sequence with decoy functionality containing CpG dinucleotides wherein cytosines are differentially modified or replaced by 5-methylcytosine or other pyrimides known in that art to cause a more potent inhibition of DNA methyltransferases is unknown. U.S. Pat. No. 5,503,975 describes the use of modified bases or nucleobases, however integration of such sequences with additional sequence elements capable of binding two or more transcription factors is unknown. The use of sequences capable of binding and or inhibiting methylated-DNA-binding proteins and or DNA methyltransferases as also integrating overhanging sequences capable of binding proteins associated with telomere maintenance is unknown. Additionally, use of polynucleic acids containing CpG dinucleotide pairs and their modification by methylation or hemimethylation within auxiliary elements as competitively capable of modulating the binding of transcription factors, serving as substrates, inhibitors, or ‘decoys’ for DNA methyltransferase (DNMT) and or methylated-DNA-binding proteins, and eliciting host innate immune responses to exposure to foreign polynucleic acids is unrealized or unknown.
The use of two or more auxiliary sequences binding transcription factors and combinations thereof as described and selected from the group of CRE/ATF-, E2F-, SP-, NFKB-, STAT-, EGR-, E-Box-elements collectively for the purposes of inhibiting cellular growth is unknown. Understanding polynucleic acid biotransformation and metabolites, use of backbone modifications in auxiliary sequences of a larger composite structures to facilitate differential persistence of such sequences for hybridization-based activities is also unknown.
To conclude, numerous events and biological activities occur upon contacting cells with polynucleic acids. Primary sequence information and structures formed by inter- and intramolecular associations are primary determinates of these biologic activities. Polynucleic acids described herein represent deliberate designs to achieve unrecognized opportunities in combining two or more polynucleic acid elements and modes of action toward enhancements of biologic activity for therapeutic benefit. In particular, this is where a multi-targeted approach will show improved efficacy and provide persistent therapy for diseases associated with cellular growth.